In many biomedical applications it is important to characterize the population of RNAs in a cell. This is useful in many research applications and clinical diagnostics. Certain quantitative genetic analyses of biological tissues and organisms are best performed at the single cell level. However, single cells only contain picograms of genetic material. Molecular protocols have been introduced to reveal the transcriptome by sequencing the RNAs of individual cells.
Microscopy, fluorescence-activated cell sorting (FACS), or real-time PCR-based methods can provide a single-cell aspect to experiments but are able to assay only a handful of genes at a time. High-throughput technologies such as microarrays and RNA-Seq provide a full view of the expression of all genes, but require more genetic material than is found in a single cell and are usually performed with thousands to millions of cells. These techniques provide useful genetic information at the cell population level, but have serious limitations for understanding biology at the single cell level.
Recently, a method, referred to as CEL-Seq protocol, was developed for overcoming the limitation of the small starting amount of RNA (Hashimshony et al., 2012, Cell Reports 2, p 666-673). The method utilizes barcoding and pooling samples before linearly amplifying mRNA with the use of one round of in vitro transcription. The described method showed more reproducible, linear, and sensitive results than a PCR-based amplification method. The robust transcriptome quantifications enabled by the method was also demonstrated to be useful for transcriptomic analyses of complex tissues containing populations of diverse cell types.
In order to identify transcriptomes, several methods that scale up have been introduced. Current biological tools also lack the capacity to assay genetic measurements in many single cells in parallel. Conventional single cell techniques are slow, tedious, and limited in the quantity of cells that can be analyzed at once. Microfluidics has been used to automate the process, achieving on the order of 100 cells per run, each very expensive. Robotics has also been developed to scale up the process to an order of magnitude of 1000 cells. However, both approaches involve the purchase of costly machines and parts, and in both of them the process is time consuming and not straight forward.
U.S. patent application No. 2014/0066318 describes methods and products for the localized or spatial detection of nucleic acid in a tissue sample. International patent applications Nos. WO 2013/180567, WO 2007/022026, WO 2010/142954, WO 2013/188872, WO 2014/201273 and U.S. patent application US 2011/0111981 relate to single cell isolation and/or analysis.
International patent applications No. WO 2015/118551 provides provided an apparatus and a method for isolation and cytometric analysis of cells from a liquid medium. The contents of WO 2015/118551 are incorporated herein by reference in their entirety.
There is a need for improved methods and systems for performing massive parallel nucleic acid analysis (e.g., transcriptome analysis) of single cells.